1. Field of the Invention
The present invention relates generally to nanoparticles that are composed of a nanocrystal core surrounded by a shell of semiconductor molecules wherein the surface of the shell is coated with a bioactivation peptide. More particularly, the present invention is directed to improving the photoluminescence and quantum yield of such peptide-coated nanoparticles.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography. The contents of the publications and other reference materials are hereby incorporated by reference.
Core/shell nanocrystals (NCs) such as CdSe/ZnS are nanometer scale inorganic clusters of semiconductor material useful for fluorescent labeling in multicolor biological imaging and detection (1, 2). These colloidal NCs consist of an inorganic particle and an organic coating that determines their solubility, functionality, and influences their photophysics. In order for these NCs to be biocompatible, they must be water-soluble, nontoxic to the cell, and offer conjugation chemistries for attaching recognition molecules to their surfaces. In addition they should efficiently target to biomolecules of interest, be chemically stable, and preserve their high photostability. The requirements for their application in single-molecule biological studies are even more stringent: fluorescent NCs should be monodisperse, have relatively small size (to limit steric hindrance), reduced blinking, large saturation intensity and high photoluminescence quantum yield (QY).
Two coating steps are necessary to render CdSe NCs synthesized in organic solvents highly luminescent, water-soluble, and biocompatible. The first coating step is the chemical deposition of higher band gap inorganic shells over NC cores (3-6). These shells serve as isolation layers, protecting the exciton wavefunction from nonradiative recombination processes at surface traps. The second coating step utilizes ligand exchange to functionalize the NCs. Various coating chemistries have been described: silanization (7, 8), mercaptoalkanoic acid ligands (9), organic dendrons (10), amphiphilic polymers (11), phospholipid micelles (12), recombinant proteins (13), and oligomeric phosphines (14). The fact that several different coatings have continuously been introduced points to the difficulty in achieving all desired properties with one universal coating. It implies that different coatings will most likely be necessary for various applications. NCs with thicker coatings will tend to have better photostabilities and higher quantum yields whereas smaller NCs with thin coatings may be less photostable but should be better suited as intracellular probes.
As set forth in PCT US2003/014401, ligand exchange of nanopartilces, such as CdSe/ZnS core/shell NCs, with phytochelatin-related peptides was found to provide bioactive NCs with only a thin water-soluble shell (15). Peptide coating endows the NCs with exceptional colloidal properties as proven by HPLC, gel electrophoresis, atomic force microscopy (AFM), transmission electron microscopy (TEM), and fluorescence antibunching studies (16). These peptides have a C-terminal adhesive hydrophobic domain with multiple cysteinyl thiolate binding sites and a hydrophilic domain that gives the NCs their desired solubility and functionality. However, this previously reported biofunctionalization scheme significantly reduces the QY of CdSe/ZnS NCs in aqueous buffer.